During pregnancy, there is a dramatic enhancement of agonist induced NO as well as PGI2 production by the uterine artery, so leading to a reduction in vascular resistance and increase in blood flow necessary to support the growing fetus. Studies comparing the effects of heptahelical receptor agonists AII and ATP vs growth factors bFGF and VEGF in P- UAEC have further revealed that while not all these agonists can mobilize Ca2+ in these cells, both heptahelical receptors and growth factor receptor activation results in activation of Extracellular signal Regulated Protein Kinase 1 and 2 (ERK-1/2) in P-UAEC, and for each class of agonist this correlates directly to their ability to stimulate NO production in particular (see below). In addition, parallel studies in NP- UAEC have shown a broad range of agonists, including ATP, AII and TPA, fail to stimulate NO production or couple efficiently to ERK-1/2 activation that otherwise couple efficiently in P-UAEC. Further identification and understanding of coupling to and function of the MAPK cell signaling pathways, and how this changes in pregnancy is the goal of this proposal. Furthermore, as yet unidentified signaling pathways may also be mediating the growth factor but not heptahelical receptor effects in NP-UAEC to produce NO, and therefore in turn that pregnancy induced changes in UAEC function may involve a possible synergy with unidentified ERK-1/2 independent pathways due to increased coupling to ERK-1/2 activation. To this end we propose: Specific Aim 1: to establish if the action of bFGF or VEGF, but not AII or ATP on MEK mediated activation of ERK-1/2 occurs via growth factor receptor autophosphorylation and subsequent phosphotyrosine recruitment of adapter proteins (Shc/Grb2/SOS) leading to Ras/Raf activation of MEK in P-UAEC and at what level this is uncoupled in NP-UAEC. Specific Aim 2: to establish if AII and ATP mediated activation of MEK occurs through alternate pathways independent of growth factor receptor phosphorylation and adapter protein recruitment, namely via the Src/Raf/MEK cascade in P-UAEC and at what level this response is uncoupled in NP-UAEC. Specific Aim 3: to further delineate the changes which occur in cell signaling in progression from the nonpregnant to pregnant state and so further clarify the mechanistic basis for these changes. This will be investigated by: (3A) further comparing the time course of activation of the Shc/Grb2/SOS/Ras/Raf pathway and the Src/Raf/MEK pathway in response to bFGF, VEGF, All, ATP and phorbol ester (TPA -control) in P-UAEC vs NP-UAEC, and (3B) to further investigate possible MEK independent signaling via P13- kinase/AKT in NP-UAEC and P-UAEC. Specific Aim 4: To establish the importance of these signaling intermediates so identified in Aims 1-3 at the level of agonist-stimulated NO production in NP-UAEC and P-UAEC by observing the effects of selective inhibitors on activity of signaling intermediates and relating any such effects to changes in NO production and eNOS phosphorylation respectively. Specific Aim 5:(5A) to relate these observed changes back to the pregnancy induced changes in vivo. Thus where signaling pathway intermediates have been shown to be lost or gained at the level of function in previous specific aims 1-4, or where mRNA encoding a signaling intermediate is altered in 5B, we will perform western analysis on UA endothelium and immunohistochemistry on UA sections from ovex, follicular phase, luteal phase and pregnant ewes, or RT-PCR quantification of UA-endothelial cell mRNA from the same animals to determine if a change in expression occurs in vivo. (5B) to use recently developed molecular techniques to investigate further possible differences in mRNA species from P-UAEC vs NP-UAEC and so identify additional signaling molecules as well as other factors which are induced and/or lost by pregnancy but may not yet be identified as such at this time. We believe these studies will provide a significant advance in both the understanding of normal control of UA endothelial function and the mechanism underlying pregnancy-induction of increased refractoriness to a variety of vasoconstrictors in pregnancy, and may also provide a new model of endothelial cell function on which future studies searching for the dysfunction leading to preeclampsia and IUGR may be based.